State the application's broad, long-term objectives and specific aims, making reference to the health relatedness of ; the project (i.e., relevance to the mission of the agency). Describe concisely the research design and methods for achieving these goals. Describe v the rationale and techniques you will use to pursue these goals. i In addition, in two or three sentences, describe in plain, lay language the relevance of this research to public health. If the application is funded, this i description, as is, will become public information. Therefore, do not include proprietary/confidential information. DO NOT EXCEED THE SPACE : PROVIDED. The metabolic syndrome has been defined clinically as a disorder characterized by dyslipidemia, hypertension, central obesity and a predisposition to premature atherosclerotic cardiovascular disease and type 2 diabetes. It is also associated with capillary rarefaction and impaired angiogenesis in muscle. In the preceding grant periods, we demonstrated that AMPK is operative in cultured vascular endothelial cells '(HUVEC, HAEC) and that its activation protects them against the apoptosis, inflammation and mitochondrial (dysfunction caused by incubation in media enriched in glucose, palmitate or TNF-alpha. In addition, we demonstrated the existence of a Sirt1/LKB1/AMPK signaling mechanism in various cultured cells and in rat liver in vivo and we have obtained preliminary evidence for its presence in cultured endothelium. In project 1, we will extend this research by carrying out studies with the following objectives: 1) To characterize the roles of Sirtl and LKB1 in mediating AMPK activation by such factors as glucose deprivation, phenformin, and resveratrol in cultured endothelial cells; 2) To determine whether hyperglycemia induces insulin resistance and apoptosis and enhances TNF-alpha - induced adhesion molecule expression in endothelial cells by causing AMPK dysregulation (decreased activity or impaired activation). If as anticipated it does, we would then determine whether this dysregulation involves inhibition pf the Sirt1/LKB1 mechanism [possibly by post-translational oxidative modification of SirT1 (with project 2)], and/or activation of protein phosphatases; and 3) To examine whether AMPK-mediated events in the vasculature are impaired in vivo in mice with endothelial-cell specific deletions of either SirT1 or LKB1. Studies will be done in cre/lox mice that will be generated by Dr. Walsh (Core B), which he will use to study ischemia-induced angiogenesis (Project 3), and Dr. Cohen (Project 2) atherogenesis in the aorta. We will characterize the effects of exercise in these and in control mice together with Dr. Nathan LeBrasseur, a rodent exercise physiologist. In particular, we will determine how SirT1 and LKB1 downregulation affect the increase in muscle capillary density and the decrease in atherogenic changes in the aorta that typically occur in response to exercise in rodents with a metabolic syndrome phenotype. j - ^These studies will provide the first information about the function of the SirT1/LKB1/AMPK signaling mechanism in the vasculature. They should also provide novel insights as to whether its dysregulation could be a cause of the endothelial cell dysfunction associated with the metabolic syndrome and a target for its therapy. ,